Fig 2. Loss of Baldspot in the Rh1^G69D eye disc alleviates ER stress and apoptosis.

(A) Degeneration caused by overexpression of Rh1^G69D is partially rescued by RNAi knockdown of Baldspot (Rh1^G69D/Baldspoti vs Rh1^G69D controls). This rescue was significant when eye size was quantified, as indicated by a larger eye in Rh1^G69D/Baldspoti flies (14043 ± 960 pixels in Rh1^G69D/Baldspoti flies and 11942 ± 473 pixels in controls). (B) Loss of Baldspot on a wild-type background had no effect on eye phenotype (26478 ± 1191 pixels and 26582 ± 1110 pixels in controls). (C) Rh1^G69D/Baldspoti eye discs display reduced apoptosis levels compared to Rh1^G69D controls, as measured by TUNEL staining. (D) Cell death was quantified by counting the number of TUNEL positive cells in each eye disc. (E) Rh1^G69D/Baldspoti eye discs had reduced expression of Xbp1-EGFP as compared to Rh1^G69D controls, while rhodopsin levels were unchanged. Eye discs were dissected from wandering L3 larvae expressing Rh1^G69D and UAS-Xbp1-EGFP, stained for rhodopsin and GFP as an indicator of ER stress, and counterstained with 4',6-diamidino-2-phenylindole (DAPI). (F) Xbp1-EGFP levels were significantly reduced in Rh1^G69D/Baldspoti eye discs (0.59 ± 0.13 fold-change from controls) as compared to Rh1G69D controls (1.00 ± 0.18). Rh1 levels were not changed (0.78 ± 0.33 fold-change from 1.00 ± 0.44 in controls). (G) Rh1^G69D/Baldspoti eye discs had reduced expression of puc-LacZ as compared to Rh1^G69D controls. (H) When quantified, LacZ levels were significantly reduced in Rh1^G69D/Baldspoti eye discs (0.16± 0.19 fold-change from controls) as compared to Rh1G69D controls (1.00 ± 0.41). (I) Rh1^G69D/Baldspoti eye discs had reduced expression of P-eif2α as compared to Rh1^G69D controls. Fluorescence was quantified using mean fluorescence from the image stack as calculated in Image J. Scale bars = 0.1 mm. * P < 0.05, ** P < 0.005, *** P < 0.0005, **** P < 0.0001.